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Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies.

ABSTRACT: Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors).We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed.The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.

SUBMITTER: Gopal H

PROVIDER: S-EPMC3378604 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Project description:BackgroundBlack flies (Diptera: Simuliidae) serve as arthropod vectors for various species of Onchocerca (Nematoda: Onchocercidae) that may be associated with disease in humans, domestic animals, and wildlife. The emergence of zoonotic Onchocerca lupi in North America and reports of cervid-associated zoonotic onchocerciasis by Onchocerca jakutensis highlight the need for increased entomological surveillance. In addition, there is mounting evidence that Onchocerca diversity in North America is far greater than previously thought, currently regarded as Onchocerca cervipedis species complex. This study reports new geographic records and black fly vector associations of an uncharacterized Onchocerca species.MethodsTo better understand the biodiversity and geographic distribution of Onchocerca, 485 female black flies (2015: 150, 2016: 335) were collected using CO2-baited traps from February to October 2015-2016 in Lake County, northern California, USA. Individual flies were morphologically identified and pooled (≤ 10 individuals) by species, collection date, and trap location. Black fly pools were processed for DNA extraction, and subsequent PCR and sequencing targeting of the NADH dehydrogenase subunit 5 gene of filarioids.ResultsAmong the pools of black flies, there were 158 individuals of Simulium tescorum (2015: 57, 2016: 101), 302 individuals of Simulium vittatum (sensu lato [s.l.]) (2015: 82, 2016: 220), 16 individuals of Simulium clarum "black" phenotype (2015: 5, 2016: 11), and 13 individuals of S. clarum "orange" phenotype (2015: 6, 2016: 7). PCR analysis revealed the percentage of filarioid-positive pools were 7.50% (n = 3) for S. tescorum, 3.75% (n = 3) for S. vittatum (s.l., likely S. tribulatum), 7.69% (n = 1) for S. clarum "black" phenotype, and no positives for S. clarum "orange" phenotype. Genetic distance and phylogenetic analyses suggest that the northern California Onchocerca isolates belong to the same species reported in black flies from southern California (average pairwise comparison: 0.32%), and seem closely related to Onchocerca isolates of white-tailed deer from upstate New York (average pairwise comparison: 2.31%).ConclusionA cryptic Onchocerca species was found in Lake County, California, and may be a part of a larger, continentally distributed species complex rather than a single described species of North America. In addition, there are at least three putative vectors of black flies (S. clarum, S. tescorum, S. vittatum) associated with this cryptic Onchocerca species. A comprehensive reassessment of North American Onchocerca biodiversity, host, and geographic range is necessary.

Project description:BackgroundThe abundance of onchocerciasis vectors affects the epidemiology of disease in Sudan, therefore, studies of vector dynamics are crucial for onchocerciasis control/elimination programs. This study aims to compare the relative abundance, monthly biting-rates (MBR) and hourly-based distribution of onchocerciasis vectors in Abu-Hamed and Galabat foci. These seasonally-based factors can be used to structure vector control efforts to reduce fly-biting rates as a component of onchocerciasis elimination programs.MethodsA cross-sectional study was conducted in four endemic villages in Abu-Hamed and Galabat foci during two non-consecutive years (2007-2008 and 2009-2010). Both adults and aquatic stages of the potential onchocerciasis vector Simulium damnosum sensu lato were collected following standard procedures during wet and dry seasons. Adult flies were collected using human landing capture for 5 days/month. The data was recorded on handheld data collection sheets to calculate the relative abundance, MBR, and hourly-based distribution associated with climatic factors. The data analysis was carried out using ANOVA and Spearman rank correlation tests.ResultsData on vector surveillance revealed higher relative abundance of S. damnosum s.l. in Abu- Hamed (39,934 flies) than Galabat (8,202 flies). In Abu-Hamed, vector populations increased in January-April then declined in June-July until they disappeared in August-October. Highest black fly density and MBR were found in March 2007 (N = 9,444, MBR = 58,552.8 bites/person/month), and March 2010 (N = 2,603, MBR = 16,138.6 bites/person/month) while none of flies were collected in August-October (MBR = 0 bites/person/month). In Galabat, vectors increased in September-December, then decreased in February-June. The highest vector density and MBR were recorded in September 2007 (N = 1,138, MBR = 6,828 bites/person/month) and September 2010 (N = 1,163, MBR = 6,978 bites/person/month), whereas, none appeared in collection from April to June. There was a significant difference in mean monthly density of S. damnosum s.l. across the two foci in 2007-2008 (df = 3, F = 3.91, P = 0.011). Minimum temperature showed significant correlation with adult flies counts in four areas sampled; the adult counts were increased in Nady village (rs = 0.799) and were decreased in Kalasecal (rs = -0.676), Gumaiza (rs = -0.585), and Hilat Khateir (rs = -0.496). Maximum temperature showed positive correlation with black fly counts only in Galabat focus. Precipitation was significantly correlated with adult flies counts in Nady village, Abu-Hamed, but no significance was found in the rest of the sampled villages in both foci. Hourly-based distribution of black flies showed a unimodal pattern in Abu-Hamed with one peak (10:00-18:00), while a bimodal pattern with two peaks (07:00-10:00) and (14:00-18:00) was exhibited in Galabat.ConclusionTransmission of onchocerciasis in both foci showed marked differences in seasonality, which may be attributed to ecology, microclimate and proximity of breeding sites to collection sites. The seasonal shifts between the two foci might be related to variations in climate zones. This information on black fly vector seasonality, ecology, distribution and biting activity has obvious implications in monitoring transmission levels to guide the national and regional onchocerciasis elimination programs in Sudan.

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Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies.

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